WO2011090653A9 - Puces à micro-groupements et procédés pour leur fabrication - Google Patents

Puces à micro-groupements et procédés pour leur fabrication Download PDF

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Publication number
WO2011090653A9
WO2011090653A9 PCT/US2010/061442 US2010061442W WO2011090653A9 WO 2011090653 A9 WO2011090653 A9 WO 2011090653A9 US 2010061442 W US2010061442 W US 2010061442W WO 2011090653 A9 WO2011090653 A9 WO 2011090653A9
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chip
substrate
microstructures
photoresist material
photoresist
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PCT/US2010/061442
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WO2011090653A1 (fr
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Jyh-Lyh Juang
Yi-You Huang
Po-Cheng Chen
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National Health Research Institutes
WANG, Lu-hai
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Priority claimed from US12/649,993 external-priority patent/US20100167950A1/en
Application filed by National Health Research Institutes, WANG, Lu-hai filed Critical National Health Research Institutes
Publication of WO2011090653A1 publication Critical patent/WO2011090653A1/fr
Publication of WO2011090653A9 publication Critical patent/WO2011090653A9/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00504—Pins
    • B01J2219/00509—Microcolumns
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/0074—Biological products
    • B01J2219/00743—Cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/12—Specific details about manufacturing devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips

Definitions

  • the present invention relates generally to microarray chips, and more specifically to microarray chips comprising concave and convex microstructures.
  • a single cell microarray system has been used for analyzing cellular response of individual cells, in which a single chip made from polystyrene contains microchambers to accommodate cells. See Yomamura et al. (2005) Anal. Chem. 77: 8050-8056.
  • Microwells of microarray systems have been fabricated from agarose, acrylamide, polydimethylsiloxane (PDMS), and etc., to confine and control cells and their growth on the surface of a substrate.
  • Conventional methods for fabricating microarray systems have involved preparing a porous substrate to increase the surface area of a microarray, and consequently the throughput capacity and sensitivity, wherein the pores serve as sites for attachment of one or more biomolecules.
  • Chin et al. used the photoresists SU-8 5 and SU-8 100 to construct microwells on a glass slide. See Chin et al. (2004) Biotechnology and Bioengineering 88 (3): 399-415. However, it had been reported that photoresist SU-8 does not adhere well to silicon dioxide. D C S Bien et al. (2003)
  • the invention relates to a three-dimensional (3-D) chip, which comprises: a) a substrate; and b) a photoresist material adhered onto one surface of the substrate, the photoresist material comprising a plurality of microstructures; wherein the substrate is one chosen from a silicon substrate and a quartz substrate.
  • the invention relates to a three-dimensional (3-D) chip, which comprises: a) substrate; and b) a polymeric material adhered onto one surface of the substrate, the polymeric material comprising a plurality of convex microstructures.
  • the invention relates to a set of paired 3-D chips, which comprises: a) a first 3-D chip; and b) a second 3-D chip.
  • the first 3-D chip in the paired set as aforementioned comprises: (i) a first substrate; and ii) a first photoresist material adhered onto one surface of the first substrate, the first photoresist material comprising a plurality of microstructures; each of the microstructure having a shape of a well.
  • the second 3-D chip in the paired set comprises: (i) a second substrate; and (ii) a second photoresist material adhered onto one surface of the second substrate, the second photoresist material comprising a plurality of microstructures, each of the microstructures having a shape of a column, the column having a size that fits into the well of the first 3-D chip.
  • the aforementioned substrate is replaced with a glass substrate with the proviso that the photoresist is not one chosen from SU-8 5, SU-8 100, and SU-8 2000 series, and wherein the photoresist material of the 3-D chip remains adhered to the glass substrate when immersed in a liquid or a culture medium for at least 3 days.
  • each of the microstructures has a diameter of > 10 micrometer but ⁇ 10,000 micrometer.
  • > 10 ⁇ but ⁇ 10,000 ⁇ it meant that all integer unit amounts within the range are specifically disclosed as part of the invention.
  • 10, 11, 12 . . . 997, 998, 999 and 10,000 ⁇ unit amounts are included as embodiment of this invention.
  • the microstructures are either concave or convex structures.
  • the photoresist material comprises an arrayed series of microstructures.
  • the photoresist material forms the microstructures, and each of the microstructures has a shape of a column.
  • the aforementioned 3-D chip may further comprise two or more than two different compounds; each of the compounds being located and/or coated on the microstructures, with each of the microstructures having a shape of a column, in a spatially discrete region of the 3-D chip.
  • the plurality of microstructures are formed through the photoresist material, and each of the microstructures has a shape of a well.
  • the well is distributed in and through the photoresist material, and surrounded by the photoresist material adhered onto one surface of the substrate, in which the bottom of the well is on the surface of the substrate.
  • the 3-D chip as aforementioned further comprises two or more than two different nucleic acids, each of the nucleic acids being located and/or coated on the microstructures (or microwells) in a spatially discrete region of the 3-D chip.
  • the photoresist material is at least one chosen from SU-8 2-15, SU-8 50-100, SU-8 2000 series, SU-8 3000 series and KMPR ® 1000 series.
  • the photoresist material remains adhered onto the surface of the substrate when immersed in a culture medium for at least three days.
  • the photoresist material remains adhered onto the surface of the substrate during air storage for more than one week.
  • each of the convex microstructures in the 3-D chip as aforementioned has a shape of a column.
  • the polymeric material comprises an arrayed series of micrcolumns.
  • the polymeric material is at least one chosen from polydimethylsiloxane (PDMS), polyethylene glycol (PEG) and polyethylene glycol diacrylate (PEGDA).
  • PDMS polydimethylsiloxane
  • PEG polyethylene glycol
  • PEGDA polyethylene glycol diacrylate
  • the 3-D chips further comprises two or more than two different compounds, each of the test compounds being located and/or coated on the
  • microstructures in a spatially discrete region of the 3-D chip are described.
  • the second photoresist material in the paired set as aforementioned is replaced with a polymeric material.
  • the second 3-D chip in the paired set further comprises two or more than two different compounds, each of the compounds being located and/coated on the microstructures in a spatially discrete region of the second 3-D chip.
  • FIG. 1 A is a scanning electron microscopic image showing 3-dimensional morphology of a microwell (concave microstructure) chip.
  • FIG. IB is a scanning electron microscopic image showing 3-dimensional morphology of a microcolumn (convex microstructure) chip.
  • FIG. 2 shows phase contrast microscopic images of HeLa cells cultured in a 2592 -well cell chip at different magnifications (40x and lOOx).
  • FIG. 3 shows fluorescent microscopic images of HeLa cells cultured in a 2592-well cell chip at different magnifications (40x and lOOx).
  • FIG. 4 is a microscopic image of HeLa cells cultured in a 40098-well cell chip.
  • FIG. 5 A is a phase contrast microscopic image of 293T cells in a 40098-well cell chip.
  • FIG. 5B is a fluorescent microscopic image of 293T cells in a 40098-well cell chip.
  • FIG. 6A is a phase contrast microscopic image of HeLa cells transfected with siGLO green transfection indicator in a 2592-well chip.
  • FIG. 6B is a fluorescent microscopic image of HeLa cells transfected with siGLO green transfection indicator in a 2592-well cell chip.
  • FIG. 7A is a fluorescent microscopic image showing TNF-oc-induced subcellular localization of NF- ⁇ in HeLa cells pretreated with PDTC.
  • FIG. 7B is a fluorescent microscopic image showing the cell nuclei of the cells in FIG. 7A.
  • FIG. 7C is a fluorescent microscopic image showing T F-a-induced subcellular localization of NF-KB in non-pretreated HeLa cells;
  • FIG. 7D is a fluorescent microscopic image showing the cell nuclei of the cells in FIG. 7C;
  • FIG. 7E is a fluorescent microscopic image showing TNF-a-induced subcellular localization of NF-KB in HeLa cells pretreated with Y294002.
  • FIG. 7F is a fluorescent microscopic image showing cell nuclei of the cells in FIG. 7E.
  • substrate shall generally mean a material having a rigid or semirigid surface or surfaces.
  • a substrate generally has top and bottom sides, and each side has a surface. The surface on one of the sides of the substrate is used for coating.
  • a substrate material includes, but not limited to, plastics plastics (e.g., polypropylene or polystyrene), ceramic, silicon, silica, glass, or quartz.
  • a substrate that is transparent to light is useful for fabricating microarray chips for assays that involve optical detections.
  • a non-transparent substrate may be used for analyses that involve non- optical detections, such as laser microarray scanner detections.
  • microcolumn chip and “drug chip” are interchangeable.
  • microwell chip and “cell chip” are interchangeable.
  • spatially discrete region shall generally mean an area on a substrate that is distinct or separate from another area on the substrate.
  • each microwell occupies a specific area on the substrate.
  • the specific areas may be distributed on the substrate in, for example, a random or uniform distribution.
  • curving shall generally mean curving inwards.
  • curving outwards shall generally mean curving outwards.
  • well shall generally mean a hole, a cavity or a space formed to contain a sample and/or a liquid.
  • column shall generally mean a rigid, relatively slender, upright support; any column like object or formation.
  • the shape of the microstructure includes but is not limited to a well (e.g. microwell), a depression, a recess, a hole, a groove, a cavity, a pit, a pore, a trench, a channel, a concaved region, a channel-connected well, and other similar shapes known to those skilled in the art.
  • a well e.g. microwell
  • the shape of the microstructure includes but is not limited to a column (e.g. microcolumn), a protrusion, a post, a hump, a hill, a ridge, a bump, a bulge, a prominence, a projection, a convex region, and other similar shapes known to those skilled in the art.
  • the microstructures may be a plurality of microwells defined on a surface of a substrate to contain or hold cells or a compound.
  • the microstructures may be a plurality of microcolumns defined on a surface of a substrate to provide areas for containing or holding probes, drugs or test compounds.
  • the microstructures may be prepared by "patterning".
  • the method of patterning includes a photolithographic exposure and development. During a photolithographic exposure, a light source of an appropriate wavelength is used to transfer a geometric pattern containing an image of desired microstructures from a photomask to a coating material adhered onto a surface of a substrate.
  • a method of patterning may comprise embossing a coating material.
  • photoresists there may be both positive and negative tone photoresists used in the photolithographic process.
  • a positive resist wherever the resist material is to be removed is exposed to UV light. The UV exposure makes the positive resist becomes more soluble in a developer. The exposed resist is washed away by a developer solution, leaving windows of bare underlying material. Therefore, the mask contains an exact copy of the pattern which is to be remained on the surface of a wafer.
  • exposure to UV light causes a negative resist to cross-link and become more difficult to dissolve. Therefore, the negative resist remains on the surface wherever it is exposed and a developer solution removes only the unexposed portions.
  • a Mask used for a negative photoresist therefore contains an inverse (or photographic "negative") of the pattern to be transferred.
  • Two different patterns of photomasks were used in fabricating 3-D chips according the invention.
  • a photomask having a pattern of a white area containing black spots was used.
  • the negative photoresist under the white area of the photomask was exposed to the light, which caused a cross-link reaction.
  • the resist after cross-linking reaction was not dissolved by a subsequent developer.
  • the resist under the black spots was unexposed to the UV, not cross-linked, and thus is dissolved by the developer, leaving holes formed in and through the resist film of the chip, i.e., resulting in microwell or cell chips.
  • a photomask having a pattern of a black area containing white spots was used.
  • the negative photoresist under the black area of the photomask was unexposed to the light, no cross-link reaction resulted and thus was dissolved by a subsequent developer.
  • the resist under the white spots was exposed to the UV, cross-linked and was thus not dissolved by the developer, resulting in convex structures formed from the resist, i.e., resulting in the formation of microcolumn or drug chips.
  • microcolumns are not limited to those made from photoresist materials. They may be fabricated from other polymers which are not photoresists by micromachined or MEMS fabrication processes, which are known in the art. For example, three dimensional microstructures, such as microcolumns may be fabricated in optical gain medium using two photon induced photopolymerization technique described by Yokoyama et al. (2003) “Thin Solid Films “ (438-439): 452-456 and Mendonca et al. (2008) Applied Physics A (90): 633-636. In the
  • femtosecond pulse laser may be used to machine any kind of material such as a metal, dielectric, semiconductor, or polymer.
  • the processing is driven by a multiphoton absorption of energy from the pulse laser, resulting in the breaking of all bonds and the atomizing of materials.
  • This laser processing is also capable of very high spatial resolution with the highest precision in the range of hundreds of nanometers.
  • a photochemical reaction is initiated through a radical mechanism following two photon excitation of a photoinitiator.
  • the photo-reactive resins that are most commonly used are acylate monomers or acrylic pre- polymers, which can be made to cross-link with the use of a radical photoinitiator molecule.
  • a polymeric material such as PDMS or PEG may be applied into the microwells of a cell chip and cover the microwell chip by forming a thin layer of polymer coat or film.
  • a substrate is then applied onto the polymer coat or film and adhere to the polymer.
  • the whole assembly containing the polymer-coated microwell chip and a substrate attached onto the polymer coat film was then treated heat for PDMS or UV for PEG.
  • the substrate and the polymer coat film adhered to the substrate can then be separated from the assembly and results in a chip containing microcolumns with a pattern that mirrors the microwell chip used at the beginning of fabrication.
  • An automated spotting device such as Perkin Elmer Biochip ArrayerTM may be used for applying a sample to a microarray chip.
  • Many contact and non-contact microarray printers are available and may be used to print a binding molecule, such as a probe, a ligand or an agonist, on a substrate.
  • non-contact printers are available from Perkin Elmer (BioChip ArrauerTM), Labcyte and IMTE (TopSpotTM). These devices utilize various approaches for non-contact spotting, including piezo electric dispension; touchless acoustic transfer; en bloc printing from multiple microchannels; and the like.
  • Other approaches include ink jet-based printing and microfluidic platforms.
  • Non-contact printing may be adopted for the production of high-specificity cellular microarrays.
  • no solid printer part contacts the array surface.
  • no aberrations or deformities are introduced onto the substrate surface, thereby preventing uneven or aberrant cellular capture at the site of spotted probe.
  • Such printing methods find particular use with high specificity hydrogel substrates.
  • the cell chips according to the invention may be used for high throughput screening for ligands that are capable of causing changes in cells.
  • ligand binding may produce a phenotypic change, such as a change in cell morphology, cell survival, apoptosis, cell migration, specific organelle, protein subcellular localization, protein level, enzyme production, enzyme activity, nucleotide level, or nucleotide subcellular localization.
  • the microarray chips according to the invention are also useful for high throughput detection of an analyte of interest in a sample using various candidate probes.
  • the candidate probes may be labeled with a detectable substance such as a fluorescent molecule, a chemiluminescent fragment, or a radioactive molecule.
  • the sample is delivered to microwells on a microarray chip according to the invention, in which probes are immobilized onto the surface of the substrate inside the microwells. Washing the microwells removes unbound sample, and the bound analyte is retained in the microwells, which can be detected, either directly or indirectly.
  • Fluorescent signals within the microwells of a microarray chip can be quantified by scanning the array with a confocal camera or with a CCD camera. Detection may also be label-free.
  • the surface Plasmon resonance or microring methods have been shown for detecting the binding of analytes to probes or the changes of cell morphology or cell volume. See Jordan et al. (1997) Anal. Chem. 69: 4939-4947, Ferreira et al. (2009) J. AM. CHEM. SOC. 131:436-437 and Peterson et al. (2009) BMC Cell Biology 10: 16.
  • Molecules or compounds may be immobilized either covalently (e.g., utilizing single reactive thiol groups of cysteine residues,) or non-covalently but specifically (e.g., via immobilized
  • the substrate should be polyfunctional or be capable of being polyfunctionalized or activated with reactive groups capable of forming a covalent bond with the target to be immobilized (e.g. carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like).
  • reactive groups capable of forming a covalent bond with the target to be immobilized (e.g. carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like).
  • a drug or a ligand may be delivered to a cell sample using microcolumns or similar structures on a array chip.
  • Microcolumns of an array chip (or a drug chip) are first applied with a drug or ligand.
  • the drug-containing microcolumns are then inserted into microwells holding or containing the cell sample to allow the release of the test drug or ligand into the microwells.
  • the microcolumn chip may be removed and microwells are washed to remove unbound drug or ligand.
  • the cellular target molecules in response to the drug or the ligand may be detected by using immunolabeling reagents and a fluorescent detector/quantifier with optical access to the microwells, either through a transparent or translucent substrate.
  • Quartz wafers (Tekstarter, Taiwan) were cleaned according to a Piranha Clean procedure (See http://engineering.tufts.edu/microfab/index_files/SOP/PirarihaClean_SOP.pdf) dried, and coated with a photoresist material. Briefly, quartz wafers were placed in a TEFLONTM carrier, submerged in a bath of 96%H 2 SO 4 :30%H 2 O 2 solution (1: 1) for 10-20 minutes to remove all organic deposits, and rinsed in deionized (DI) water for 15 minute. The cleaned quartz wafers were blown dry with nitrogen or dried in an oven at 120°C or on a hotplate at 150°C and placed in a carrier box until ready for coating.
  • DI deionized
  • photoresist material such as SU-8 100 (MicroChem, Newton MA) was spin-coated on quartz wafers as follow. Wafers were subjected to a spread cycle, during which the wafers were ramped to 500 rpm at an acceleration of 100 rpm/second, and held at 500 rpm for 10 seconds to allow the resist to cover the entire surface on one side of the wafer. The wafers then were subjected to a spin cycle, during which the wafers were ramped to 3000 rpm at an acceleration of 300 rpm/second and held at 3000 rpm for 30 seconds to obtain a photoresist coat or film having a thickness of about 100 ⁇ m. Changing the conditions of the spread and/or spin cycles would have impacts on the thickness of the photoresist coat/film on the wafers. For example, an increasing in the speed at a spin cycle would reduce the thickness of the resist coat/film on the wafers.
  • SU-8 100 MicroChem, Newton MA
  • photoresist-coated wafers were soft baked on a hot plate at 65°C for 10 minutes and then at 95 °C for 30 minutes.
  • a standard for photoresist coat/film on the wafers was
  • FIG. 1 shows an electron microscopic image of a microarray chip having a plurality of wells (with each having a diameter of -500 ⁇ ) defined on a quartz wafer.
  • Glass wafers were cleaned according to a Piranha clean procedure as described above, dried and coated with a photoresist. Briefly, quartz wafers were placed in a TEFLONTM carrier, submerged in a bath of 96%H 2 SO 4 :30%H 2 O 2 solution ( 1 : 1 ) for 10-20 minutes to remove all organic deposits, and rinsed in deionized (DI) water for 15 minute. ) After the Piranha Clean the glass wafer was blown dry with nitrogen and stored in a carrier box until ready for coating.
  • DI deionized
  • photoresist such as SU-8 3050 (MicroChem, Newton MA) was spin- coated on glass wafers as described below. Briefly, during a spread cycle the wafers were ramped to 500 rpm at an acceleration of 100 rpm/second and held at 500 rpm for 10 seconds to allow the resist to uniformly coat on the surface on one side of each wafer. At a spin cycle, the wafers were ramped to 1000 rpm at an acceleration of 300 rpm/second and held at 1000 rpm for 30 seconds to obtain a photoresist coat or film having a thickness of about 100 ⁇ . The wafers were soft baked on a hot plate at 95 °C for 45 minutes.
  • the photolithography process was performed by using an UV light source.
  • the EVG 620 Top Side Mask Aligner was used to align patterns on wafers to expose the resist at 375 mJ/cm 2 and UV below 350nm was eliminated.
  • the SU-8 3050 film was baked on a hot plate at 65 °C for 1 minute, 95 °C for 5 minutes.
  • the patterns were developed with SU-8 developer (MicroChem, Newton MA) for 15 minutes.
  • the SU-8 3050 film was rinsed with isopropyl alcohol, air-dried with nitrogen, and subjected to hard baking on a conventional oven at 150 °C for 15 min.
  • Silicon wafers were cleaned according to a Piranha clean procedure as described above, dried, and coated with a photoresist. Briefly, silicon wafers placed in a TEFLONTM carrier were submerged in H 2 0:H 2 0 2 :NH 4 OH solution (5:1 : 1) for 10 minutes, rinsed in deionized (DI) water for 1 minute, submerged in H 2 0:HF solution (50: 1) for 15 seconds, rinsed in DI water for 1 minute, submerged in H 2 0:H 2 0 2 :HCl solution (6: 1 : 1) for 10 minutes and rinsed in DI water for 1 minute. The silicon wafers were blown dry with nitrogen and placed in a carrier box until ready for coating.
  • DI deionized
  • SU-8 50 film (MicroChem, Newton MA) was spin-coated on silicon wafers as follow. During a spread cycle wafers were ramped to 500 rpm at an acceleration of 100 rpm/second and held at 500 rpm for 10 seconds to allow the resist to coat the surface on one side of each wafer. At a spin cycle, the wafer was ramped to 2000 rpm at an acceleration of 300 rpm/second and held at 2000 rpm for 30 seconds to obtain a photoresist coat or film having a thickness of having a thickness of about 50 ⁇ . The wafers were soft baked on a hot plate at 65°C for 6 minutes and 95 °C for 20 minutes.
  • the SU-8 50 film was rinsed with isopropyl alcohol, air-dried with nitrogen and subjected to hard baking on a conventional oven at 150 °C for 15 min. As a result, a drug array chip having a plurality of columns (each having a diameter of about 350 ⁇ ) defined on the silicon wafer was formed.
  • NANOTM SU-8 100 was used to fabricate microwells on the glass substrate of the microarray chip. See Chin et al. (2004) Biotechnology and Bioengineering 88 (3): 399-415.
  • NANOTM SU-8 2-25, NANOTM SU-8 50-100 photoresist and SU-8 2000 series such as NANOTM SU-8 2000.5-2015, NANOTM SU-8 2025-2075 or NANOTM SU-8 2100-2150 film, provided very weak adhesion to glass substrate.
  • photoresist coat/film might peel off from the surface of the glass substrate when microarray chips were stocked in the air after one week at room temperature or immersed for in the culture medium during cell culture.
  • the resist SU-8 100 peeled off the grass substrate after a week (i.e., the stability lasted for only a week), and the resist SU-8 2000 peeled off the glass substrate after 3 days.
  • the resist SU-8 100 peeled off the glass slide within 2-3 days, and SU 8 2000 peeled off the glass slide within one day.
  • Various photoresist materials such as SU-8 100, SU-8 2050 and SU-8 3050, MPR 1050 films, were tested for their adhesion against a set of substrates, such as silicon, quartz and glass substrates, using an adhesion tester (ROMULUS III universal tester) at National Nano Device
  • NDL Laboratory
  • Aluminum nails were affixed onto a photoresist test film that was adhered onto a substrate.
  • the tested film was baked on a hot plate at 150°C for an hour to allow the aluminum nails to adhere to the tested film, cooled down and mounted on a clamping device.
  • a breaking point platform having a force system and force transducer was included in the adhesion tester to provide a 0 kg to 100 kg downward pulling force.
  • the adhesion tester was semi-automated by a computer workstation to measure the maximal adhesion of the film. Any cracking on the tested film was checked to determine if the testing results were positive. Positive test results were recorded (Table 1). Otherwise, the test would be repeated on another test film.
  • photoresist films tested except KMPR 1050 were found to peel off from the glass substrate as soon as it was fabricated thereon even before the adhesion test was conducted. By contrast, both silicon and quartz substrates have good adhesion with most of the photoresist films tested.
  • Adhesion tests were also conducted on fabricated chips in a cell culture environment. Briefly, the photoresist film coated on the surface of the glass or quartz wafer was defined and patterned by a photolithpgraphic process to form a plurality of microwells on the wafer. The wafer was diced using a dicing saw (a precision dicing system) into chips of a standard microscope slide size (75mm x 25mm). After storage for about 2 weeks, the fabricated chips were sterilized and placed in culture dishes (10 cm diameter each). The Minimum Essential Medium (MEM) was added into the dishes and incubated at 37°C. The fabricated chips in the culture dishes were examined with naked eyes 2 days after the incubation, and results are shown in Table 2.
  • MEM Minimum Essential Medium
  • silicon and quartz substrates had good adhesion to all the photoresist tested and thus can provide a stable environment for cell microarray chips.
  • the glass substrate had poor adhesion to both photoresit SU-8 100 and SU-8 2050.
  • the photoresist SU-8 100 film and photoresist SU-8 3050 film showed similar adhesion to glass in the physical adhesion test (Table 1), however, the microwells constructed through the SU-8 100 film were found to peel off from the glass surface during the cell culture. Only the microwells constructed through the photoresist SU-8 3050 were intact, adhering to the surface of the glass for long term maintenance of cell culture in the microarray chip (Table 2).
  • HeLa cells Human cervical cancer cell line
  • 293T cells Human embryonic kidney cell line commonly used for transfection assay were selected for transfection experiments.
  • the HeLa cells were grown in MEM and 293T cells grown in Dulbecco's Modified Eagle's Medium at 37°C in a 5% C0 2 incubator. Both MEM and DMEM were supplemented with 10% FCS and 100 units/ml penicillin/streptomycin.
  • the cultured cells were collected as cell suspensions.
  • the HeLa cells were transferred to each microwell of a 2,592-well or 40,098-well cell chips fabricated according to Examples 1 or 2.
  • the 293T cells were transferred to each microwell of a 40,098-well microarray cell chip.
  • the living cells were observed under a phase contrast microscope and the images shown are shown in FIG. 2 and 4, 5A.
  • Cells were also labeled with fluorescent cell markers and detected using a fluorescent confocal microscope and the images are shown in FIGs. 3 (HeLa cells) and 5B (293T cells).
  • the HeLa cells were transfected with siGLO green transfection indicator according to the manufacturer's instruction (Dharmacon Inc.). Briefly, fluorescent RNA duplexes (siRNA) were spotted into a 2,592-well microarray cell chip at 0.001 pmole/well. Next, 1,575 ⁇ L of rehydration solution (25 uL of DharmaFECT and 1,550 ⁇ L of RNase-free water) was dispensed onto the chip. The DharmaFECT transfection reagent was allowed to complex with the RNA duplexes (siRNA) at room temperature for 20 minutes.
  • siGLO green transfection indicator according to the manufacturer's instruction (Dharmacon Inc.). Briefly, fluorescent RNA duplexes (siRNA) were spotted into a 2,592-well microarray cell chip at 0.001 pmole/well. Next, 1,575 ⁇ L of rehydration solution (25 uL of DharmaFECT and 1,550 ⁇ L of RNase-free water) was
  • the cell chip having microwells containing siGLO green transfection indicator was transferred into a culture dish, to which 12mL of cell suspension (6.25 x 10 5 cells per mL) were added. The cells were incubated at 37°C, 5% CO 2 to allow transfection to occur and observed at 48 h posttransfection using a phase contrast microscope (FIG. 6A) and a fluorescent microscope (FIG. 6B).
  • FIG. IB a cell chip
  • FIG. 1C a drug chip
  • the cell chip comprises an arrayed series of thousands of microwells
  • the drug chip comprises an arrayed series of thousands of microcolumns.
  • the microcolumns of the drug chip were complementary to the microwells of the cell chip in terms of the arrayed pattern and size.
  • the size of each mcirocolumn is no larger than each microwell in the diameter and the height.
  • HeLa cells were seeded in the microwells (60 x 10 4 cells/well) of a 2,666-well cell chip (each well diameter and depth:— 500 ⁇ m by—l00 ⁇ m) and cultured overnight at 37 °C.
  • the drug chip was pre-treated with 0.01% poly-L-lysine (PLL) (Sigma) to coat the surfaces of the microcolumns formed from the photoresist material.
  • PLL poly-L-lysine
  • Different concentrations of test drugs were individually spotted onto the microcolumns of a drug chip.
  • PDTC pyrrolidine dithiocarbamate
  • Alginate was used to absorb/retain the drug PDTC. In some experiments, gelatin instead of alginate was used.
  • the drug/alginate mixtures were individually spotted onto the top of the microcolumns of a drug chip (each microcolumn diameter and height: ⁇ 350 ⁇ m by ⁇ 50 ⁇ m) using a pipetman.
  • phosphoinositide 3- kinase inhibitor (LY294002) was spotted onto different rows of microcolumns of the drug chip.
  • the final concentrations of all of the drugs applied onto the microcolumns were 2mM.
  • the drug chip was then placed at 4 °C overnight to dry the drug mixtures.
  • the drug chip was inserted into the microwells of the cell chip with the drug spots on the microcolumns facing down and entering the openings of the cell-containing microwells of the cell chip.
  • the entire assembly of the drug chip and cell chip was placed in an incubator with humidity > 95% at 37°C for 4 hours, during which the PDTC or LY294002 was released from the microcolumns and treated the cells in the microwells.
  • the drug chip was then removed afterwards.
  • the cell chip was rinsed 3 times to remove the residual PDTC or LY294002 in the culture medium, followed by incubation of cells with TNF-a for 30 minutes.
  • TNF-a has been known to induce NF- ⁇ to translocate from the cytoplasm to the nucleus.
  • Immunoassay was performed to determine subcellular localization of NF- ⁇ as follows.
  • the cells on the cell chip were washed with PBS (10ml) in a dish (10 cm), fixed with paraformaldehyde (3.7%, 2ml) at room temperature for 20 minutes, washed twice with PBS (10ml).
  • the cells were blocked with BSA (1%, 10 ml) at room temperature for 30 minutes, incubated with the primary antibody NF- ⁇ antibody at room temperature for 1 hour, washed three times with PBS (10ml, 5min each) and then incubated with a secondary antibody conjugated with tetramethyl rhodamine iso- thiocyanate (Rhodamine (TRITC)-conjugated anti-rabbit secondary antibody), and 4'-6-diamidino-2- phenylindole (DAPI) at room temperature for 1 hour in the dark.
  • the cells were washed three times with PBS (10ml) for 10 minutes each.
  • FIG. 7 top panel shows a cell chip containing an arrayed series of thousands of microwells, in which the regions labeled (i)-(iii) contained microwells having cell cultures that were pretreated with PDTC prior to TNF-a treatment.
  • the regions labeled (iv)-(vi) contained microwells having cell cultures that were pretreated with LY294002 prior to TNF-a treatment.
  • the area between the region (iii) and the region (iv) contained microwells having cell cultures that were not pretreated any drug prior to the exposure to TNF-a.
  • FIGs. 7A, 7C, 7E show TRITC staining of cells for NF- ⁇
  • FIGs. 7B, 7D, 7F show corresponding DAPI staining for the cell nucleus.
  • PDTC-pretreated cells (7A, 7B) in microwells from the region (i) showed a significantly less induction of nuclear localization of NF- ⁇ by TNF-a, as compared to the non-drug-pretreated cells in microwells from the region between (iii) and (iv) (7C, 7D).

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Abstract

L'invention porte sur des puces à micro-groupements en trois dimensions (3-D). La puce à micro-groupements en trois dimensions comprend un substrat et un matériau de résine photosensible qui est mis sous forme d'un motif afin de comprendre une pluralité de microstructures, par exemple des micro-puits ou des micro-colonnes. L'invention porte également sur des procédés de fabrication et d'utilisation de celles-ci. Les puces à micro-groupements en trois dimensions trouvent une utilisation particulière dans des analyses à haut rendement de production.
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